GB1588054A - Preparation of d-n-benzyloxycarbonyl-2-(p-hydroxyphenyl) glycine and l-p-hydroxyphenyl-glycine from racemic n-benzyloxycarbonyl-2-(p-hydroxyphenyl) glycine by treatment with an actinomycete - Google Patents

Description

(54) PREPARATION OF D-N-BENZYLOXYCARBONYL-2-(P-HYDROXY- PHENYL)GLYCINE AND L-P-HYDROXYPHENYL-GLYCINE FROM RACEMIC N-BENZYLOXYCARBONYL-2-(P-HYDROXYPHENYL)GLYCINE BY TREATMENT WITH AN ACTINOMYCETE (71) We, F. HOFFMANN-LA ROCHE & CO., AKTIENGESELLSCHAFT, a Swiss Company of 124 - 184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention is concerned with a process for the cleavage of a racemate, namely of DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine.

The process provided by the present invention comprises treating DL-N benzyloxycarbonyl-2-(p-hydroxyphenyl-glycine with an Actinomycete belonging to the genus Streptomyces and capable of effecting the asymmetric cleavage of this racemate and subsequently isolating the resulting D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine and L-p-hydroxyphenyl-glycine from the mixture obtained.

The'D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine obtainable in accordance with the present invention is a valuable intermediate for the manufacture of semi-synthetic penicillins and cephalosporins. This optically active intermediate has hitherto been produced by cleavage of the corresponding racemate using a cleavage agent, the optically active isomers being obtained by fractionation of the corresponding diastereomers and treatment of the products obtained with an acid or base. Japanese Kokai (published specification) No. 69039/75 discloses such a method using (+)-a-phenylthylamine as the cleavage agent and a similar process is described in German Offenlegungsschrift No. 1 942 693 using quinine as the cleavage agent. In comparison with the hitherto known processes, the process provided by the present invention is significantly simpler and cheaper.

The microorganism used in the present process can be any Actinomycete which belongs to the genus Streptomyces and which is capable of effecting the cleavage of racemic N-benzyloxycarbonyl-2-(p-hydroxyphenyl )-glycine. The microorganism can be used, for example, in the form of a culture broth or in the form of an extract thereof.

Preferred strains used in the present process are derived from an Actinomycete belonging to the genus Streptomyces which has been isolated from a soil sample in Seacliff Park, Adelaide City, South Australia. Australia. Also preferred are analogous strains as well as mutants and variants thereof. The aforementioned isolated Actinomycete has been deposited in Fermentation Research Institute. Agency of Industrial Science and Technolo gy. Japan. as "FERM-P No. 3666". A subculture of this deposited Actinomycete has been deposited in the microorganism collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, USA. under No. NRRL 11057.

The characteristic mycological features of the Actinomycete deposited as FERM-P No.

3666 and NRRL 11057 are as follows: 1. Morphologz: The strain forms a well developed aerial mycelium (0.3 - 0.8 x 1.0 - 1.4 a) with straight to slightly curved spore-forming hyphae. Neither curled nor spiral formation is observable.

Spores, which appear in straight to slightly curved chains with more than 50 spores per chain, are cylindrical with smooth surface.

2. Growth of the various media: The following colour tones are given in accordance with Color Harmony Manual, 4th Edition, 1958 (Publisher: Container Corporation of America, Chicago, USA).

1) Saccharose nitrate agar (cultivated at 270C): The growth is good, colourless to brownish-grey. The aerial mycelium is greyish-white to light brownish grey (3dc, Natural).

No soluble pigment is produced.

2) Glucose/asparagine agar (cultivated at 27"C): The growth is good, colourless to slight yellow. The aerial mycelium is greyish-white to light brownish-grey. Slight production of a pale-yellowish, soluble pigment.

3) Glycerine/asparagine agar (ISP-5, cultivated at 27"C): The growth is slight olive coloured (1 1/2 ge, Light Olive Gray) to pale yellowish-brown (2gc, Bamboo). The aerial mycelium is yellowish-white (2ba, Pearl) to light brownish-grey 3foe, Silver Gray). Slight formation of a pale-yellow, soluble pigment.

4) Inorganic salts/starch agar (ISP-4, cultivated at 27"C): The growth is pale yellowish-brow.n (2tug, Mustard Tan) to dark olive coloured. The aerial mycelium is light brownish-grey (3dc, Natural) to light greyish-reddish-brown (5fe, Ashes). The lower side is pale yellowish-brown to dark olive coloured. Slight production of a yellowish, soluble pigment.

5) Tyrosine agar (ISP-7, cultivated at 27"C): The growth is pale yellowish-brown (2ie, Light Mustard Tan) to dark yellowish-brown. The aerial mycelium is light grey to brownish-grey (3fe, Silver Gray). Slight formation of a yellowish, soluble pigment.

6) Nutrient agar (Waxmans N-agar, cultivated at 27 C): The growth is colourless to pale yellowish-brown. The aerial mycelium is greyish-white to light brownish-grey. Slight production of a brownish, soluble pigment.

7) Yeast/malt agar (ISP-2, cultivated at 27"C): The growth is yellowish-brown (3pi, Golden Brown). The aerial mycelium is light brownish-grey (3dc, Natural) to light greyish-reddish-brown (5fe, Ashes). The lower side is yellowish-brown to dark yellowishbrown. No soluble pigment.

8) Oatmeal agar (ISP-3, cultivated at 27"C): The growth is light olive-grey (1 1/2 ie, Light Olive) to pale-yellow (2ec, Oat Meal). The aerial mycelium is light brownish-grey (3ec, Light Beige) to light greyish-reddish-brown (5fe, Ashes). The lower side is light olive-grey to pale yellow. A yellow, soluble pigment is produced.

9) Glocuse/peptone/gelatine stab culture (cultivated at 25DC): The growth is colourless to slight yellowish-brown. No aerial mycelium is produced, but a dark brown, soluble pigment.

10) Skimmed milk (cultivated at 370C): The growth is pale-yellow, the aerial mycelium white. Brownish, soluble pigment is produced.

3. Physiological properties: 1) Optimal growth temperature: On yeast/malt agar no growth is observable at 100C and 45"C, whereas slight to abundant growth takes place at 25"C. 27 C. 3ü C and 37"C. The optimal growth temperatures lies at about 27"C.

2) Gelatine liquefaction on glucose/peptone/gelatine agar (cultivated at 25"C): The liquefaction is positive, but of slight to average intensity.

3) Hydrolysis of the starch of inorganic salts/starch agar (cultivated at 270C): The hydrolysis is positive, but of average intensity.

4) Coagulation and peptonisation in 1()% skimmed milk (cultivated at 37"C): The peptonisation is positive (average to strong), the coagulation positive (average).

5) Melanin formation at 27"C: No pigment on tyrosine agar (1SP-7). but brown-black pigment not only in tryptone (yeast broth (lSP-l) but also on peptone/yeast/iron agar (ISP-6).

6) Utilisation of carbohydrates on Pridhan-Gottlieb agar (ISP-9), (cultivated at 27"C): Abundant growth with D-xylose. D-glucose and L-rhamnose. Good growth with L-arabinose. Slight growth with D-fructose. No growth with Saccharose. inositol. raffinose and D-mannitol as well as with the controls (no addition).

The following is a compilation of the aforementioned mycological properties of the strain FERM-P No. 3666 (NRRL 1 1(157): FERM-P No. 3666 (NRRL 11()57) is an Actinomycete belonging to the genus Streptomyces having straight to slightly curved aerial mycelium with neither curled nor spiral formation being present. In various culture media there is produced pale-yellow to yellowish vegetative mycelium which forms light brownish-grey to light greyish-reddish brown aerial mycelium. On some agar culture media there are found soluble pigments with yellow colour tones. Melanin pigments are formed not only in tryptone/yeast broth (ISP-1) but also on peptone/yeast/iron agar (ISP-7). Hydrolysis of average intensity of starch and protein is observable.

Known microorganisms having properties closely related to the strain FERM-P No. 3666 (NRRL 11057) are Streptomyces xanthocidicus (International Journal of Systematic Bacteriology, volume 22, page 372, 1972, and Journal of Antibiotics Ser. A. volume 19, pages 195-199, 1966) and Streptomyces zaomyceticus (International Journal of Systematic Bacteriology, volume 22, page 374, 1972 and Journal of Antibiotics, Ser. A, volume 7, pages 134-136, 1954). The following Table shows a comparison between these three microorganisms: TABLE Streptomyces Streptomyces FERM-P No. 3666 zaomyceticus xanthocidicus Properties (NRRL 11057) ISP-5196 ISP-5575 Morphology of the aerial straight straight stright mycelium slightly curved slightly curved slightly curved Inorganic salts/starch agar; light brownish-grey light brownish-grey light brownish grey aerial mycelium light greyish- (thin) light greyish -reddish-brown -reddish-brown Growth pale-yellowish-brown pale yellowish-brown pale yellowish-brown dark olive-coloured yelow-brown Soluble pigment slightly yellow slightly yellow slightly yellow On oatmeal agar; aerial light brownish-grey light brownish-grey light brownish-grey mycelium light greyish- light greyish- light greyish -reddish-brown -reddish-brown -reddish-brown Growth light olive-grey pale yellowish-brown pale yellow olivepale-yellow olive brown -grey Soluble pigment yellow yellow none On glucose/peptone/gelatine dark brown dark brown none or slight agar; soluble pigment Melanin formation on ISP-1 + + + ISP-6 + + + ISP-7 - - (-) TABLE (continued) Streptomyces Streptomyces FERM-P No. 3666 zaomyceticus xanthocidicus Properties (NRRL 11057) ISP-5196 ISP-5575 Reduction of nitrate + + Milk coagulation - -#(+ at 37 C) Milk liquefaction ++ + Gelatine liquefaction + + + Starch hydrolysis + + + Utilisation of carbohydrates L-arabinose + + ++ D-xylose ++ + ++ D-glucose ++ ++ ++ D-fructose # - ++ saccharose - - ++ inositol - - L-Rhamnose ++ - Raffinose - - + D-Manitol - - (++ abundant growth; + good growth; # slight growth) The foregoing comparisons show the similarities between the strain FERM-P No. 3666 (NRRL 11057), Streptomyces zaomyceticus and Streptomyces xanthocidicus. FERM-P No.

3666 (NRRL 11057) differs, however, from Streptomyces xanthocidicus in its capability of reducing nitrate, in the utilisation of saccharose, L-rhamnose and raffinose and in the capability of coagulating skimmed milk. FERM-P No. 3666 (NRRL 11057) differs from Streptomyces zaomyceticus in the form of the aerial mycelium on inorganic salts/starch agar and in the utilisation of L-rhamnose. On the other hand, in its morphology of the aerial mycelium, in its growth characteristics on glucose/peptone/gelatine medium and in its other physiological properties, the strain FERM-P No. 3666 NRRL 11057) is more closely related to Streptomyces zaomyceticus than to Streptomyces xantho.cidicus. The strain FERM-P No. 3666 (NRRL 11057) has been named Streptomyces zaomyceticus NRJA28-C MYla.

The DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine used as the starting material in the process provided by the present invention can be prepared according to a number of known methods; for example, by reacting DL-p-hydroxyphenyl-glycine with benzyloxycar bonyl chloride.

The process provided by the present invention can be carried out by inoculating a culture medium containing DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl) -glycine with spores or mycelia of the Actinomycete to be used. The cultivation conditions are not particularly limited, although it is preferred to carry out the cultivation in an aqueous medium, which contains not only carbon and nitrogen sources but also inorganic salts, under aerobic conditions (which are brought about, for example, by shaking). Carbon sources are, for example, glucose and maltose, nitrogen sources are, for example, polypeptones, yeast extract, meat extract and cornsteep liquor and inorganic salts are, for example, phosphates or manganese,copper zinc or iron salts. The addition of special growth factors such as, for example, amino acids, nucleosides, vitamins or blood serum, is not necessary.

The temperature at which the cultivation is carried out generally lies in the range of from 20 to 40"C, preferably from 25"C to 35"C. The cultivation is preferably carried out for 3-7 days. The cultivation is preferably carried out at a pH of 5.0-9.0.

During the asymmetric hydrolysis of the DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl) glycine in accordance with the present invention the pH rises; at the end of the cultivation it usually lies at about 8.8.

The mixture obtained after the cultivation contains D-N-benzyloxycarbonyl-2-(p hydroxyphenyl)-glycine and L-p-hydroxyphenyl-glycine. These two optically active com pounds can be isolated from the culture medium according to methods known per se. For example, the mycelium is removed from the culture broth by centrifugation or filtration, the culture solution obtained is acidified (e.g. with a mineral acid) and D-N benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine is subsequently extracted from the acidi fied solution with a water-immiscible organic solvent such as, for example, ethyl acetate or butyl acetate. By removal of the solvent under reduced pressure there are obtained impure crystals of D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine. Optically pure D-N benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine is obtained by purification of the crude crystals by means of usual purification methods such as recrystallisation or chromatog raphy.

The aqueous phase which contains L-p-hydroxyphenyl-glycine can be conducted through a column filled with a strong acidic cation exchange resin. the column being eluted with aqueous ammonia. The eluate is concentrated and subsequently conducted through a column filled with weak acidic cation exchange resin. By eluting the column with water there is obtained a concentrated liquid containing L-p-hydroxyphenyl-glycine which is evaporated under reduced pressure. Optically pure L-p-hydroxyphenyl-glycine is obtained as a colourless crystallisate after treatment of the residue with organic solvent such as ethanol.

The process provided by the present invention yields optically pure D-N benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine and L-p-hydroxyphenyl-glycine in high yields in a technically simple manner.

The following Examples illustrate the present invention: Example 1 Three 50() ml Erlenmeyer flasks are each charged with a solution (pH 7.0) of 9()() mg of DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine in 300 ml of aqueous medium containing 2% maltose. 0.5%' polypeptone. 0.5coo meat extract. 0.3% yeast extract, 0.3coo sodium chloride. 0.1% magnesium sulphate. 80 ppm divalent manganese ions, 70 ppm divalent copper ions. 2() ppm divalent zinc ions and 10 ppm divalent iron ions. and the content of the flasks is subsequently sterilised. After the steriiisation, each flask is inoculated with the strain FERM-P No. 3666 (NRRL 11057) of the microorganism Streptomyces zaomyceticus NRJA28-C-MYla. The thus-obtained culture is incubated for 5 days at 270C on a rotary shaking apparatus at 180 revolutions per minute.

The resulting fermentation solutions (pH 8.8) are combined and centrifuged to remove the mycelium. The supernatant phase is adjusted to pH 2.0 with concentrated aqueous hydrochloric acid and subsequently extracted three times with 300 ml of ethyl acetate each time. The organic phase is separated, dried over anhydrous sodium sulphate and evaporated under reduced pressure to a crystalline residue. Recrystallisation of the residue from chloroform containing a trace of methanol yields 340 mg of colourless needle-like crystals of D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine [yield based on the original amount of D-enantiomer: 75.5%; melting point: 157 -160 C; [a]i',3 = -118" (c = 1 in methanol)].

The aqueous phase remaining after extraction with ethyl acetate is filtered through a column filled with Dowex 50 (H-form, 43 ml; trade mark of Dow Chemicals USA). The column is washed with 150 ml of water and subsequently eluted with 0.3% aqueous ammonia. The fractions which are positive to ninhydrin are combined and concentrated to about 3 ml under reduced pressure. The concentrate is conducted through a column filled with Amberlite CG-50 (Type I, 65 ml, mixed resin consisting of 7 parts by volume of ammonium form and 3 parts by volume of the H-form of the resin; trade mark of Rhm and Haas) and the column is eluted with water. The fractions which are positive to ninhydrin are combined and evaporated under reduced pressure. Crystallisation of the residue from ethanol yields 185 mg of L-p-hydroxyphenyl-glycine in the form of colourless crystals [yield based on the original amount of L-enantiomer: 74.1%; melting point: 212"-214"C; [a]i',3 = +108 (c = 1 in water).

Example 2 A fermentation is carried out in the manner described in Example 1 with the exception that 300 mg of DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine are used and that the fermentation time amounts to 6 days.

After the fermentation. the mycelium is centrifuged off and the supernatant phase (pH 8.8) adjusted to pH 2.0 with concentrated aqueous hydrochloric acid. The aqueous solution is extracted twice with 70 ml of ethyl acetate each time. The organic extracts are combined, dried over aqueous sodium sulphate and concentrated under reduced pressure. The concentrate is added to a column filled with 20 ml of Wacogel C-200 (trade mark-Waco Junyaku Kogyo K.K.) and equilibrated with chloroform. The column is eluted with a mixture of chloroform and ethanol (20:1). The fractions which are positive to the Pauli reaction are combined and concentrated under reduced pressure to a crystalline residue.

Recrystallisation of the residue from chloroform containing a small amount of methanol yields 123 mg of D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine [yield: 82%; melting point: 1590-1600C; [a]Dt = -123 (c = 1 in methanol)l.

The aqueous phase remaining after extraction with ethyl acetate is worked-up in the manner described in Example 1, there being obtained 67 mg of crystalline L-phydroxyphenyl-glycine [yield: 80.50/c: melting point: 212"-215"C; [a]D" = -109.5" (c = 1 in water)].

Claims (12)

WHAT WE CLAIM IS:

1. A process for the cleavage of a racemate, which process comprises treating DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine with an Actinomycete belonging to the genus Streptomyces and capable of effecting the asymmetric cleavage of this racemate and subsequently isolating the resulting D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)- glycine and L-p-hydroxyphenylglycine from the mixture obtained.

2. A process according to claim 1, wherein an Actinomycete belonging to the species Streptomyces zaomyceticus is used.

3. A process according to claim 2. wherein Streptomyces zaomyceticus FERM-P- No.

3666 (NRRL 11057) is used.

4. A process according to any one of claims 1 to 3 inclusive. wherein the treatment is carried out in an aqueous nutrient medium which contains carbon and nitrogen sources and inorganic salts.

5. A process according to any one of claims 1 to 4 inclusive. wherein the treatment is carried out under aerobic conditions.

6. A process according to any one of claims 1 to 5 inclusive. wherein the treatment is carried out at a temperature in the range of from 20"C to 4() C.

7. A process according to claim (z. wherein the treatment is carried out at a temperature in the range of from 25"C to 35 C.

8. A process according to any one of claims l to 7 inclusive. wherein the treatment is carried out at a pH of 5.()-9.tl.

9. A process according to any one of claims 1 to 8 inclusive. wherein the D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine is separated from the L-phydroxyphenyl-glycine by removing the mycelium from the culture broth, acidifying the culture solution obtained and extracting the D-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)- glycine from the acidified solution with a water-immiscible organic solvent.

10. A process for the cleavage of DL-N-benzyloxycarbonyl-2-(p-hydroxyphenyl)glycine, substantially as hereinbefore described with reference to Example 1 or Example 2.

11. D-N-Benzyloxycarbonyl-2-(p-hydroxyphenyl)-glycine, when prepared by the process claimed in any one of claims 1 to 10 inclusive or by an obvious equivalent thereof.

12. L-p-Hydroxyphenyl-glycine, when prepared by the process claimed in any one of claims 1 to 8 inclusive or claim 10 or by an obvious equivalent thereof.